Outer membrane vesicles and uses thereof

ABSTRACT

The disclosure relates to outer membrane vesicles from  Francisella  and  Piscirickettsia , and their use in vaccine compositions. In particular, the present disclosure relates to compositions and methods useful in inducing protective immunity against francisellosis or salmon rickettsial septicaemia (SRS) in fish.

FIELD OF THE INVENTION

The disclosure relates to outer membrane vesicles from microorganisms,and their use in vaccine compositions. In particular the disclosurerelates to outer membrane vesicles from Francisella and Piscirickettsia,and their use in vaccine compositions. The disclosure relates to outermembrane vesicles from Francisella and Piscirickettsia, and their use invaccine compositions. In particular, the present disclosure relates tocompositions and methods useful in inducing protective immunity againstfrancisellosis or salmon rickettsial septicaemia (SRS) in fish.

BACKGROUND OF THE INVENTION

Francisella species (spp) are non-motile, pleomorphic, gram-negative,strictly aerobic, facultative intracellular coccobacilli. They areextremely infectious, as less than 10 bacteria are required forinfection (Jones et al., 2005; Soto et al., 2009; Kamaishi et al.,2010). One member of the genus, Francisella noatunensis, has beenreported worldwide as a cause of francisellosis in fish (Kamaishi etal., 2005; Mauel et al., 2005; Olsen et al., 2006; Mauel et al., 2007;Birkbeck et al., 2007; Jefferery et al., 2010). F. noatunensis iscomposed of two subspecies adapted to different host temperatures, oneof which (F. noatunensis ssp. orientalis) causes disease in fish livingin warmer waters (Kamaishi et al., 2005; Mauel et al., 2005; Mauel etal., 2007; Jeffery et al., 2010) while the second (F. noatunensis ssp.noatunensis) causes disease in fish living in colder waters (Nylund etal., 2006; Olsen et al., 2006; Birkbeck et al., 2007). Outbreaks offrancisellosis in fish aquaculture can be devastating, causing largelosses worldwide (Kamaishi et al., 2005; Mauel et al., 2005; Olsen etal., 2006; Mauel et al., 2007; Birkbeck et al., 2007; Jefferery et al.,2010), and represents the main challenge for aquaculture based onAtlantic cod Gadus morhua L. It is also sporadically problematic inaquaculture base on Tilapia, one of the largest produced fish worldwide.

Piscirickettsia salmonis is described as non-motile, not-encapsulated,pleomorphic coccoid, with a size ranging from 0.1-1.5 um (Mauel andMiller, 2002, Vet Microbiol, 87:279-289). Salmon Rickettsial Septicaemia(SRS), caused by P. salmonis, is a disease of salmonid fish with a hugeimpact on the salmonid fish farming particularly in Chile. Similarly toFrancisella sp., also P. salmonis is intracellular in nature makingvaccine development challenging. The mortality rate of affected fishvaries, from more than 90% mortality in some Chilean outbreaks, to lowlevels of mortality in e.g. Norway. The reason for the observeddifferences in mortality is not known, and although environmentalfactors must be taken into account, strain difference is also likely. Asannual losses due to SRS in Chile are estimated to be more than 200million USD each year, the potential impact on the salmon aquaculturecould be devastating. Despite the availability of several commercialvaccines against SRS with reported good efficacy in laboratory trials(Wilhelm et al., 2006, Vaccine, 24:5083-5091; list of available vaccinesare reviewed in the Australian Aquatic Veterinary Emergency Plan,Disease Strategy Piscirickettsiosis, 2013), SRS was reported asresponsible for 60% of the mortality of salmon and 79% of the mortalityrainbow trout in Chile in 2011 (Integrated Annual and SustainabilityReport 2012, Cermaq: EWOS Innovation-SRS project in Chile). Clearly,there is a demand for a vaccine against SRS with improved efficacy. Upuntil recently, one of the main challenges within P. salmonis researchand vaccine development has been the lack of growth of the pathogen inliquid culture media. Yañez et al., (2012), reported the AUSTRAL-SRSbroth, a highly complex medium consisting of a marine-based brothsupplemented with L-cysteine, that supported the growth of P. salmonisreaching an optical density of approx OD_(600 nm)=1.8 after 6 daysincubation. Improvements of growth rate and increased biomass was madeby growth in basal medium 3 (BM3) reaching an OD_(600 nm) of 1.7 after37.5 hrs (Henriquez et al., 2013). BM3 consist of yeast extract (Merck)2.0 g L21, peptone from meat (peptic digested, Merck) 2.0 g/L,MgSO4*7H2O 0.1 g/L, K2HPO4 6.3 g/L, NaCl 9.0 g/L, CaCl2*2H2O 0.08 g/L,FeSO4*7H2O 0.02 g/L and glutamic acid 2 g/L.

Aquaculture is able to prevent outbreaks of many bacterial infectionsthat presented huge problems for the industry in its youth, by the useof vaccines composed of inactivated in vitro cultured whole-cellbacterial preparations (bacterins) supplemented with adjuvants (reviewedby Brudeseth et al., 2013). As a consequence of this, the use ofantimicrobials in Norwegian aquaculture has declined enormously despitea large increase in the amount of fish produced (reviewed by Sommersetet al., 2005). No commercial vaccine for fish francisellosis iscurrently available (reviewed by Colquhoun & Duodu, 2011; reviewed byBrudeseth et al., 2013), as attempts at using whole-cell preparations ofF. noatunensis ssp. noatunensis has yielded none or unsatisfactorylevels of protection (reviewed by Colquhoun & Duodu, 2011). This issimilar to the situation for tularemia in humans, where vaccination withkilled bacteria induces an antibody response with only limitedprotective properties (reviewed by Cowley and Elkins, 2011). The reasonfor this is due to the fact that to develop proper protection againstFrancisella spp. there is a need to stimulate cell-mediated immunity(reviewed by Cowley and Elkins, 2011), which vaccines based on killedwhole-cells or protein subunits generally are poor at (reviewed byTitball, 2008). Live attenuated vaccines (LAVs) are efficient atinducing cell-mediated immunity, though there are safety concerns suchas reversion to virulence and safety for immune-compromised individualsfor such vaccines (reviewed by Titball, 2008). Particularly in anaquaculture setting, spread of genetically modified organisms to theenvironment is another factor to take into account. A LAV designatedLive Vaccine Strain (LVS) has successfully been utilized to protecthigh-risk groups against tularemia (reviewed by Conlan & Oyston, 2007),demonstrating that it is possible to generate successful LAVs againstFrancisella spp. Several targeted deletion strains have also been shownto be protective against tularemia, such as the F. tularensis ssp.tularensis Schu S4 Δftt_0918, Δftt_0918ΔcapB and ΔclpB (reviewed byConlan & Oyston, 2007, Conlan et al., 2010). Additionally, a LAV basedon a ΔiglC mutant of F. noatunensis ssp. orientalis has recently beenpatented (U.S. Pat. No. 8,147,820 B2) for use against francisellosis inaquaculture, and has been shown to protect tilapia against experimentalchallenge (Soto et al., 2011). Previous ΔiglC mutants have been shown toinduce protective immunity in mice for F. tularensis ssp. novicida butnot against F. tularensis ssp. tularensis (Twine et al., 2005; Pammit etal., 2006). Protection obtained by vaccination with both F. tularensisssp. novicida and F. noatunensis ssp. orientalis ΔiglC mutants in miceand tilapia respectively could partly be transferred by passiveimmunization of naïve animals (Pammit et al., 2006; Soto et al., 2011).

When constructing vaccines for immunization of Atlantic cod there arecertain peculiarities of the cod immune system that should be addressed.Vaccination results in production of lower levels of specific antibodiesand less variety in the utilization of immunoglobulin heavy chain types,but despite this Atlantic cod develop protective immunity aftervaccination with most bacterial pathogens (reviewed by Samuelsen et al.,2006). The reason for this was for a long time unclear, howeverdifficulties with identifying MHC class II and associated genesindicated changes in how Atlantic cod process classical MHC class IIdependent antigens. Recently whole-genome sequencing revealed that thegenome of Atlantic cod lack MHC class II and Invariant chain (Ii), andthat CD4 is only present as a truncated pseudogene (Star et al., 2011).This would render the MHC class II antigen presenting pathway(Mantegazza et al., 2013) non-functional, and would explain the lack ofspecific antigen-responses when vaccinating with bacterins. Atlantic codhas expanded its repertoire of MHC class I antigens which mightfacilitate cross-presentation of traditional MHC class II antigens byMHC class I molecules, and there is evidence that Atlantic cod might becompensating for the loss of CD4⁺ T-cells as well by having differentsubsets of CD8⁺ T-cells (Star et al., 2011). Atlantic cod also has highlevels of natural antibodies compared to other fish species (reviewed byPilström et al., 2005), which might compensate for a strong specificantibody response on encounter with a pathogen. However, there arereports of Atlantic cod producing specific antibodies in response tovaccination with Aeromonas salmonicida, Listonella (Vibrio) anguillarumand F. noatunensis (Lund et al., 2006; Lund et al., 2007; Schrøder etal., 2009), though as they seem to be predominately recognizing LPS aT-cell independent antibody response (reviewed by Alugupalli, 2008)could explain the observed production of antibodies in response to thesebacterial pathogens.

The production of membrane vesicles by cells is a conserved mechanismoccurring throughout all domains of life, both prokaryotic andeukaryotic (reviewed by Deatherage & Cookson, 2012). In bacteria, thesevesicles are usually called Outer Membrane Vesicles (OMVs) and areformed by budding from the outer bacterial membrane (from Gram negativebacteria). They are 10-300 nm in diameter and spherical, containingouter membrane and periplasmic proteins, and recent data indicates thatthey might contain inner membrane and cytoplasmic proteins as well, andin some cases DNA (Pèrez-Cruz et al., 2013+++). The protein content ofOMVs show specific packaging, as some proteins are enriched and some areexcluded (e.g. Galka et al., 2011; Haurat et al., 2011 og mange flere).The exact sorting mechanism responsible for enrichment or exclusion ofproteins from OMVs is not currently known. Many pathogenic bacteriaincorporate virulence factors, including toxins, into their OMVs,turning the vesicles into bacterial-derived bombs (Kuehn & Kesty, 2005;Galka et al., 2011; Haurat et al., and etec+salmonella as well). OMVshave recently received renewed focus in the field of vaccinology(reviewed by Collins, 2011), as they present antigens in their nativeconformation and does not require adjuvants to be immunogenic.Immunization of humans using OMVs have been performed with great successagainst Neisseria meningitidis type B (reviewed by Granoff, 2010;Collins, 2011). OMVs derived from other bacteria have also shownprotective efficacy when used as vaccines against other pathogenicbacteria, such as Burkholderia pseudomallei (Nieves et al., 2011),Brucella melitensis (Avila-Calderòn et al., 2012), Edwardsiella tarda(Park et al., 2011), enterotoxigenic Escherichia coli (Roy et al.,2011), Salmonella Typhimurium (Alaniz et al., 2007), Shigella flexneri(Camacho et al., 2011; Camacho et al., 2013) and Vibrio cholera (Schildet al., 2008). OMVs have been shown to induce both B- and T-cellresponses (Alaniz et al., 2007. Romeu et al., 2013++)

F. tularensis ssp. has previously been shown to produce vesicles in invitro cultured infected macrophages (Anthony et al., 1991; Golovliov etal., 2003). Recent work has shown that similar vesicles could beisolated from broth cultured F. tularensis ssp. novicida and F.philomiragia ssp. philomiragia (Pierson et al., 2011), and that thesevesicles were derived by budding from the outer bacterial membrane(McCaig et al., 2013) thereby being true OMVs. Macrophages treated withthe vesicles released proinflammatory cytokines, and mice vaccinatedwith OMVs were protected against subsequent challenge with F. tularensisssp. novicida (Pierson et al., 2011; McCaig et al., 2013).Interestingly, in addition to regular spherical vesicles, OMVs from F.tularensis can also be shaped like tubes (McCaig et al., 2013).Previously, Bakkemo et al., (2011) showed by EM that also F. noatunensisssp. noatunensis releases vesicles in vitro in infected macrophages, butas they could not detect vesicles from extracellular cultured bacteria,they hypothesized that the formation of vesicles from F. noatunensisssp. noatunensis was an intracellular event.

Systems and methods for protecting fish against infection by infectiousagents are needed.

SUMMARY OF THE INVENTION

The disclosure relates to outer membrane vesicles from microorganisms,and their use in vaccine compositions in fish. In particular thedisclosure relates to outer membrane vesicles from Francisella andPiscirickettsia, and their use in vaccine compositions. In particular,the present disclosure relates to compositions and methods useful ininducing protective immunity against francisellosis or salmonrickettsial septicaemia (SRS) in fish.

Accordingly, in some embodiments, the present invention provides methodsand uses of inducing immunity against francisellosis (e.g., preventingor treating francisellosis disease and/or SRS) in a fish, comprising:administering a composition comprising an outer membrane vesicle of aFrancisella spp. or a Piscirickettsia spp. to a fish. The presentinvention is not limited to a particular species of Francisella orPiscirickettsia. Examples include, but are not limited to, Francisellanoatunensis (e.g., including but not limited to. Francisella noatunensissubsp. noatunensis; Francisella noatunensis supsp. orientalis; F.noatunensis subsp. endociliophora; or Candidatus F. noatunensis subsp.endociliophora); Francisella phlomiragia subsp. philomiragia;Francisella cantonensis; Francisella victoria; (Schrallhammer M et al.,2011); Francisella cantonensis; or Piscirickettsia spp. (e.g., includingbut not limited to, Piscirickettsia salmonis strains LF-89; (U36941);EM-90 (U36940); NOR-92 (U36942); ATL-4-91 (U36915); IRE-99D (AY498637);SCO-95A (AY498636); SCO-02A (AY498635); IRE-98A (AY498634); IRE-91A(AY498633); WSB-98; or AL10005). The uses and methods described hereinfind use in preventing and treating infection and disease in a varietyof fish species. Examples include, but are not limited to, Atlantic cod,Gadus morhua; tilapia, Oreochromis sp.; Atlantic salmon, Salmo salar;trout, Oncorhynchus mykiss hybrid striped bass, Morone chrysops×M.saxatilis or three-lined grunt, Parapristipoma trilinineatum.

Additional embodiments are described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Coomassie Blue staining of proteins from OMVs isolated fromF. noatunensis ssp. noatunensis.

FIG. 2 shows Coomassie Blue staining of proteins from OMVs isolated fromP. salmonis

FIG. 3 shows AFM of OMV isolated from P. salmonis.

FIG. 4 shows AFM and TEM on isolated OMVs from F. noatunensis ssp.noatunensis.

FIG. 5 shows TEM images of zebrafish embryos infected with F.n.n.-GFPrevealing production of OMVs in vivo. A) TEM image of an infectedzebrafish embryo revealing bacteria surrounded by OMVs in anintracellular compartment. B) TEM image of zebrafish embryo cellsheavily infected with bacteria, autophagy of cellular organelles (openarrowhead) indicating apoptosis. C) TEM image of extracellular bacteriasurrounded with OMVs in an infected zebrafish embryo. D) In vivo buddingof OMV in the cytoplasm of an infected zebrafish embryo. Asterisksindicate F.n.n., arrows indicate OMVs, arrowhead indicate formation ofOMV, open arrowhead indicate autophagic organelle.

FIG. 6 shows that F. noatunensis ssp. noatunensis causes dose-dependentmortality of adult zebrafish.

FIG. 7 shows that OMVs are safe for immunization of zebrafish andprotect against subsequent challenge with F. noatunensis ssp.noatunensis.

FIGS. 8 a, b, c, and d shows quantification of bacterial burden in thekidney, spleen and heart of unvaccinated and vaccinated fish.

FIG. 9 shows growth curves of P. salmonis in Eugon broth supplementedwith Casamino acids.

FIGS. 10 a, b, c and d provide bar graphs of levels of expression ofvarious genes in the specified organs of zebrafish infected with P.salmonis.

FIGS. 11 a, b, c and d provide bar graphs of levels of expression ofvarious genes in the specified organs of zebrafish infected with P.salmonis.

FIGS. 12 a, b, c and d provide bar graphs of levels of expression ofvarious genes in the specified organs of zebrafish injected with OMVsisolated from P. salmonis.

DEFINITIONS

As used herein, the term “under conditions such that said subjectgenerates an immune response” refers to any qualitative or quantitativeinduction, generation, and/or stimulation of an immune response (e.g.,innate or acquired).

A used herein, the term “immune response” refers to a response by theimmune system of a subject. For example, immune responses include, butare not limited to, a detectable alteration (e.g., increase) in Tollreceptor activation, lymphokine (e.g., cytokine (e.g., Th1 or Th2 typecytokines) or chemokine) expression and/or secretion, macrophageactivation, dendritic cell activation, T cell activation (e.g., CD4+ orCD8+ T cells), NK cell activation, and/or B cell activation (e.g.,antibody generation and/or secretion). Additional examples of immuneresponses include binding of an immunogen (e.g., antigen (e.g.,immunogenic polypeptide)) to an MHC molecule and inducing a cytotoxic Tlymphocyte (“CTL”) response, inducing a B cell response (e.g., antibodyproduction), and/or T-helper lymphocyte response, and/or a delayed typehypersensitivity (DTH) response against the antigen from which theimmunogenic polypeptide is derived, expansion (e.g., growth of apopulation of cells) of cells of the immune system (e.g., T cells, Bcells (e.g., of any stage of development (e.g., plasma cells), andincreased processing and presentation of antigen by antigen presentingcells. An immune response may be to immunogens that the subject's immunesystem recognizes as foreign (e.g., non-self antigens frommicroorganisms (e.g., pathogens), or self-antigens recognized asforeign). Thus, it is to be understood that, as used herein, “immuneresponse” refers to any type of immune response, including, but notlimited to, innate immune responses (e.g., activation of Toll receptorsignaling cascade) cell-mediated immune responses (e.g., responsesmediated by T cells (e.g., antigen-specific T cells) and non-specificcells of the immune system) and humoral immune responses (e.g.,responses mediated by B cells (e.g., via generation and secretion ofantibodies into the plasma, lymph, and/or tissue fluids). The term“immune response” is meant to encompass all aspects of the capability ofa subject's immune system to respond to antigens and/or immunogens(e.g., both the initial response to an immunogen (e.g., a pathogen) aswell as acquired (e.g., memory) responses that are a result of anadaptive immune response).

As used herein, the term “immunity” refers to protection from disease(e.g., preventing or attenuating (e.g., suppression) of a sign, symptomor condition of the disease) upon exposure to a microorganism (e.g.,pathogen) capable of causing the disease. Immunity can be innate (e.g.,non-adaptive (e.g., non-acquired) immune responses that exist in theabsence of a previous exposure to an antigen) and/or acquired (e.g.,immune responses that are mediated by B and T cells following a previousexposure to antigen (e.g., that exhibit increased specificity andreactivity to the antigen)).

As used herein, the term “immunogen” refers to an agent (e.g., amicroorganism (e.g., bacterium, virus or fungus) and/or portion orcomponent thereof (e.g., a protein antigen)) that is capable ofeliciting an immune response in a subject. In some embodiments,immunogens elicit immunity against the immunogen (e.g., microorganism(e.g., pathogen or a pathogen product)).

The term “test compound” refers to any chemical entity, pharmaceutical,drug, and the like that can be used to treat or prevent a disease,illness, sickness, or disorder of bodily function, or otherwise alterthe physiological or cellular status of a sample. Test compoundscomprise both known and potential therapeutic compounds. A test compoundcan be determined to be therapeutic by screening using the screeningmethods of the present invention. A “known therapeutic compound” refersto a therapeutic compound that has been shown (e.g., through animaltrials or prior experience with administration to humans) to beeffective in such treatment or prevention.

The term “sample” as used herein is used in its broadest sense. As usedherein, the term “sample” is used in its broadest sense. In one sense itcan refer to a tissue sample. In another sense, it is meant to include aspecimen or culture obtained from any source, as well as biological.Biological samples may be obtained from animals (including humans) andencompass fluids, solids, tissues, and gases. Biological samplesinclude, but are not limited to blood products, such as plasma, serumand the like. These examples are not to be construed as limiting thesample types applicable to the present invention. A sample suspected ofcontaining a human chromosome or sequences associated with a humanchromosome may comprise a cell, chromosomes isolated from a cell (e.g.,a spread of metaphase chromosomes), genomic DNA (in solution or bound toa solid support such as for Southern blot analysis), RNA (in solution orbound to a solid support such as for Northern blot analysis), cDNA (insolution or bound to a solid support) and the like. A sample suspectedof containing a protein may comprise a cell, a portion of a tissue, anextract containing one or more proteins and the like.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure relates to outer membrane vesicles from Francisella andPiscirickettsia, and their use in vaccine compositions. In particular,the present disclosure relates to compositions and methods useful ininducing protective immunity against francisellosis or salmonrickettsial septicaemia (SRS) in fish.

Vaccinating against intracellular pathogens has always been a challenge,especially without resorting to the use of live-attenuated vaccines(Titball, 2008).

Although antibodies typically have been thought to exclusively combatpathogens in the extracellular stages of infection, new informationregarding the importance of antibody-mediated immunity againstintracellular pathogens question this long-standing dogma (Casadevall &Pirofsky, 2006). But even though it has been proven that Francisella hasa significant extracellular phase where bacteria would be accessible toantibodies (Forestal et al., 2007; Yu et al., 2008), and that antibodiesagainst the LPS-O-antigen complex can award some protection againstinfection with Francisella (Sebastian et al., 2007; Cole et al., 2009),the importance of stimulating cell-mediated immunity for combatingFrancisella-infections is unquestionable. Zebrafish have been usedextensively in recent years for studying host-pathogen interactions dueto several reasons, such as easy housing, a fully sequenced genome andavailability of genetic tools (Allen & Neely, 2010). Additionally,zebrafish have a fully functional immune system remarkably similar tomammals (Meeker & Trede 2008; Renshaw & Trede, 2012). Recently, Vojtechet al. (2009) showed that adult zebrafish were susceptible to infectionwith F. noatunensis ssp. orientalis. Zebrafish embryos are susceptibleto F. noatunensis ssp. orientalis, and additionally to F. noatunensisssp. noatunensis and F. tularensis ssp. novicida when the embryos wereadapted to the proper temperatures for the pathogen (Brudal et al.,Infect Immun. 2014 June; 82(6):2180-94). In experiments associated withthe present invention, we investigated whether adult zebrafish could beinfected with F. noatunensis ssp. noatuenensis, and if zebrafish couldbe used to study the efficacy of OMVs as a vaccine againstfrancisellosis. When infecting zebrafish with F. noatunensis ssp.noatunensis, a clear dose-response with regards to mortality wasobserved, and the onset of mortality was also earlier for the groupinfected with the highest dose. After an initial acute phase ofinfection, no more mortality could be observed, and the infectionentered a chronic state. While the bacterial burden declined throughoutthe experiment, large amounts of bacteria were present in all examinedtissues even at 4 weeks post infection.

It is contemplated that vaccination with OMVs can protect zebrafishagainst development of francisellosis. This was apparent fromquantification of bacterial burden in tissues from infected fish, andalso by a clear clinical improvement for vaccinated fish compared tounvaccinated fish.

The infectious dose was chosen due to a desire to mimic thespontaneously occurring disease as much as possible. As francisellosisin Atlantic cod is a chronic, granulomatous disease (Nylund et al.,2006; Olsen et al., 2006), we wanted to avoid an acute model ofinfection.

OMVs isolated from most bacteria require detergent-extraction to reducethe levels of LPS and LOS (lipo-oligo-saccharides) to be safe forparenteral delivery (Collins, 2011). Detergent-extracted OMVs, as wellas OMVs induced by other chemical methods such as gentamicin treatment,differ in protein content compared to native OMVs (Collins 2011; van deWaterbeemd et al., 2013). As Francisella spp. contains a unique LPSwhich is less toxic compared to most Gram negative bacteria (Gunn &Ernst, 2007), native OMVs can be isolated and used for vaccinationwithout adverse effects. Recent studies using F. tularensis ssp.novicida OMVs used intra-nasal (i.n.) immunization with native vesicles(Pierson et al., 2011; McCaig et al., 2013). Here we show that zebrafishcan be immunized with native OMVs intraperitoneally (i.p.) derived fromF. noatunensis ssp. noatunensis without adverse effects.

The present invention relates to compositions, such as vaccines, andtheir use to elicit immune responses against Francisella andPiscirickettsia spp, especially protective immune responses in aquaticspecies including fish. The invention also relates to isolated outermembrane vesicles from strains of Francisella and Piscirickettsia sppwhich infect fish. It also relates to a method of preventing infectionby Francisella and Piscirickettsia spp in fish comprising administeringa vaccine of the invention, e.g., wherein said animal is a human. Itfurther relates to a method for reducing Francisella and Piscirickettsiaspp infection symptoms in fish, comprising administering to a fish orpopulation of fish in need of such treatment an effective amount ofantibodies against native outer membrane vesicles of Francisella in apharmaceutically acceptable excipient or feed vehicle, or a method ofeliciting an immune response against Francisella and/or Piscirickettsiaspp comprising administering outer membrane vesicles from Francisellaand/or Piscirickettsia spp. This invention in one aspect providesisolated or biologically pure (e.g., substantially free of toxiccomponents) outer membrane vesicles from Francisella and Piscirickettsiaspp which have been shown to infect fish as described in more detailherein. “Isolated” in general refers to the vesicles in a state otherthan the natural state. The invention also provides a vesiclepreparation. For administration to a fish, the vesicles are preferablyformulated as immunogenic compositions, and more preferably ascompositions suitable for use as a vaccine in fish. Vaccines of theinvention may either be prophylactic (i.e. to prevent infection) ortherapeutic (i.e. to treat disease after infection), but will typicallybe prophylactic.

Immunogenic compositions comprise an immunologically effective amount ofantigen, as well as any other compatible components, as needed. By“immunologically effective amount” is meant that the administration ofthat amount to an subject, such as a fish, either in a single dose or aspart of a series, is effective for treatment or prevention. This amountvaries depending upon the health and physical condition of the subjectto be treated, age, the taxonomic group or subject to be treated (e.g.,fish, etc.), the capacity of the individual's immune system tosynthesize antibodies, the degree of protection desired, the formulationof the vaccine, and other relevant factors. It is expected that theamount will fall in a relatively broad range that can be determinedthrough routine trials. Dosage treatment may be a single dose scheduleor a multiple dose schedule (e.g., including booster doses). The vaccinemay be administered in conjunction with other immunoregulatory agents.

Accordingly, embodiments of the present invention provide compositionsand methods for immunizing fish against francisellosis. In someembodiments, the present invention provides OMV isolated from a varietyof species of Francisella. The present invention is not limited to aparticular species or strain of Francisella. Examples include, but arenot limited to:

-   -   Francisella noatunensis subsp noatunensis (syn. Francisella        piscicida, syn Francisella philomiragia subsp noatunensis, syn        Francisella piscicida);    -   Francisella noatunensis supsp. orientalis (syn. Francisella        noatunensis subsp asiatica, syn Francisella victoria)        Francisella philomiragia subsp. Philomiragia; F. noatunensis        subsp. Endociliophora;    -   Wolbachia persica;    -   Francisella victoria; (Kay W, Petersen B O, Duus JØ, Perry M B,        Vinogradov E. Characterization of the lipopolysaccharide and        beta-glucan of the fish pathogen Francisella victoria. FEBS J.        2006 July; 273(13):3002-13. Epub 2006 Jun. 5.-Noda H, Munderloh        U G, Kurtti T J. Endosymbionts of ticks and their relationship        to Wolbachia spp. and tick-borne pathogens of humans and        animals. Appl Environ Microbiol. 1997 October; 63(10):3926-32);    -   Candidatus F. noatunensis subsp. endociliophora (Schrallhammer M        et al., 2011);    -   Francisella cantonensis Isolated from air conditioning systems        (Qu P, Deng X, Zhang J, Chen J, Zhang J, Zhang Q, Xiao Y,        Chen S. Identification and characterization of the Francisella        sp. strain 08HL01032 isolated in air condition systems. Wei        sheng wu xue bao Acta microbiologica sinica. 2009;        49:1003-1110); and    -   Fangia hongkongensis (Lau K W, Ren J, Fung M C, Woo P C, Yuen K        Y, Chan K K, Qian P Y, Wong P K, Wu M. Fangia hongkongensis gen.        nov., sp. nov., a novel gammaproteobacterium of the order        Thiotrichales isolated from coastal seawater of Hong Kong. Int J        Syst Evol Microbiol. 2007 November; 57(Pt 11):2665-9).

It should be noted that there are many synonyms for these species andthey have been isolated from ornamental fish to fish from marine, fresh,brackish, warm and cold water (See e.g., Colquhoun D J, Duodu S.Francisella infections in farmed and wild aquatic organisms. Vet Res.2011 Mar. 8; 42(1):47. doi: 10.1186/1297-9716-42-47).

In some embodiments, the present disclosure provides OMV isolated fromPiscirickettsia salmonis. Examples include, but are not limited to,isolates from Atlantic salmon (Salmon salar), coho salmon (Oncorhynchuskisutch) and Rainbow trout (O. mykiss). Other Rickettsia Like Organisms(RLOs) have been reported worldwide in different fish species (Khoo etal. 1995, Chen et al. 2000a,b). In some embodiments, the following P.salmonis strains are utilized:

-   -   LF-89 (ATCC VR-1361) (-Gómez F A, Tobar J A, Henríquez V, Sola        M, Altamirano C, Marshall S H. Evidence of the presence of a        functional Dot/Icm type IV-B secretion system in the fish        bacterial pathogen Piscirickettsia salmonis. PLoS One. 2013;        8(1):e54934. doi: 10.1371/journal.pone.0054934. Epub 2013 Jan.        28; Fryer J L, Lannan C N, Giovannoni S J, Wood N D.        Piscirickettsia salmonis gen. nov., sp. nov., the causative        agent of an epizootic disease in salmonid fishes. Int J Syst        Bacteriol. 1992 January; 42(1):120-6);    -   (U36941)    -   EM-90 (U36940)    -   NOR-92 (U36942)    -   ATL-4-91 (U36915)    -   IRE-99D (AY498637)    -   SCO-95A (AY498636)    -   SCO-02A (AY498635)    -   IRE-98A (AY498634)    -   IRE-91A (AY498633)    -   WSB-98 isolate of Piscirickettsia salmonis originally isolated        from liver tissue of moribund white seabass (Chen et al.        2000); P. salmonis AL10005, PHARMAQ AS, 0213 Oslo, Norway        (Eliassen, T. M.; Solbakk, I. T.; Haugseth, K. T.; Bordevik, M.;        Nygaard, A.; Rode, M. UK. Patent 2,452,892, 2011) or isolated        described McCarthy U, Steiropoulos N A, Thompson K D, Adams A,        Ellis A E, Ferguson H W (Confirmation of Piscirickettsia        salmonis as a pathogen in European sea bass Dicentrarchus labrax        and phylogenetic comparison with salmonid strains. Dis Aquat        Organ. 2005 Apr. 18; 64(2):107-19).

In some embodiments, the present invention provides vaccine compositionscomprising an OMV isolated from Francisella or Piscirickettsia species.In some embodiments, OMVs are administered alone or in a physiologicallyacceptable carrier. Physiologically acceptable carriers for vaccinationof fish are known in the art and need not be further described herein.In addition to being physiologically acceptable to the fish the carriermust not interfere with the immunological response elicited by thevaccine and/or with the expression of its polypeptide product.

The vaccines of the present invention are preferably administered tofish to prevent, e.g., francisellosis or Salmon Rickettsial Septicaemia(SRS), anytime before or after hatching. The term “fish” is defined toinclude but not be limited to fish species including trout, salmon,carp, perch, pike, eels, and char as well as mollusks and crustaceans.The vaccine may be provided in a sterile container in unit form or inother amounts. It is preferably stored frozen, below −20.degree. C., andmore preferably below −70.degree. C. It is thawed prior to use, and maybe refrozen immediately thereafter.

In some embodiments, vaccine compositions comprise one or moreadditional agents including but are not limited to, adjuvants,surfactants, additives, buffers, solubilizers, chelators, oils, salts,therapeutic agents, drugs, bioactive agents, antibacterials, andantimicrobial agents (e.g., antibiotics, antivirals, etc.). In someembodiments, a vaccine composition comprises an agent and/or co-factorthat enhance the ability of the immunogen to induce an immune response(e.g., an adjuvant). In some preferred embodiments, the presence of oneor more co-factors or agents reduces the amount of immunogen requiredfor induction of an immune response (e.g., a protective immune respone(e.g., protective immunization)). In some embodiments, the presence ofone or more co-factors or agents can be used to skew the immune responsetowards a cellular (e.g., T cell mediated) or humoral (e.g., antibodymediated) immune response. The present invention is not limited by thetype of co-factor or agent used in a therapeutic agent of the presentinvention.

Adjuvants are described in general in Vaccine Design—the Subunit andAdjuvant Approach, edited by Powell and Newman, Plenum Press, New York,1995. The present invention is not limited by the type of adjuvantutilized (e.g., for use in a composition (e.g., pharmaceuticalcomposition). For example, in some embodiments, suitable adjuvantsinclude an aluminium salt such as aluminium hydroxide gel (alum) oraluminium phosphate. In some embodiments, an adjuvant may be a salt ofcalcium, iron or zinc, or may be an insoluble suspension of acylatedtyrosine, or acylated sugars, cationically or anionically derivatisedpolysaccharides, or polyphosphazenes. In some embodiments, adjuvants aremineral oil or Montanide ISA711.

In general, an immune response is generated to an antigen through theinteraction of the antigen with the cells of the immune system. Immuneresponses may be broadly categorized into two categories: humoral andcell mediated immune responses (e.g., traditionally characterized byantibody and cellular effector mechanisms of protection, respectively).These categories of response have been termed Th1-type responses(cell-mediated response), and Th2-type immune responses (humoralresponse).

Stimulation of an immune response can result from a direct or indirectresponse of a cell or component of the immune system to an intervention(e.g., exposure to an immunogen). Immune responses can be measured inmany ways including activation, proliferation or differentiation ofcells of the immune system (e.g., B cells, T cells, dendritic cells,APCs, macrophages, NK cells, NKT cells etc.); up-regulated ordown-regulated expression of markers and cytokines; stimulation of IgA,IgM, or IgG titer; splenomegaly (including increased spleencellularity); hyperplasia and mixed cellular infiltrates in variousorgans. Other responses, cells, and components of the immune system thatcan be assessed with respect to immune stimulation are known in the art.

In some embodiments, an immunogenic oligonucleotide containingunmethylated CpG dinucleotides (“CpG”) is used as an adjuvant. CpG is anabbreviation for cytosine-guanosine dinucleotide motifs present in DNA.CpG is known in the art as being an adjuvant when administered by bothsystemic and mucosal routes (See, e.g., WO 96/02555, EP 468520, Davis etal., J. Immunol, 1998, 160(2):870-876; McCluskie and Davis, J. Immunol.,1998, 161(9):4463-6; and U.S. Pat. App. No. 20050238660, each of whichis hereby incorporated by reference in its entirety). For example, insome embodiments, the immunostimulatory sequence isPurine-Purine-C-G-pyrimidine-pyrimidine; wherein the CG motif is notmethylated.

In some embodiments, adjuvants such as Complete Freunds Adjuvant andIncomplete Freunds Adjuvant, cytokines (e.g., interleukins (e.g., IL-2,IFN-γ, IL-4, etc.), macrophage colony stimulating factor, tumor necrosisfactor, etc.), detoxified mutants of a bacterial ADP-ribosylating toxinsuch as a cholera toxin (CT), a pertussis toxin (PT), or an E. coliheat-labile toxin (LT), particularly LT-K63 (where lysine is substitutedfor the wild-type amino acid at position 63) LT-R72 (where arginine issubstituted for the wild-type amino acid at position 72), CT-S109 (whereserine is substituted for the wild-type amino acid at position 109), andPT-K9/G129 (where lysine is substituted for the wild-type amino acid atposition 9 and glycine substituted at position 129) (See, e.g.,WO93/13202 and WO92/19265, each of which is hereby incorporated byreference), and other immunogenic substances (e.g., that enhance theeffectiveness of a composition of the present invention) are used with acomposition comprising an immunogen of the present invention.

Additional examples of adjuvants that find use in the present inventioninclude poly(di(carboxylatophenoxy)phosphazene (PCPP polymer; VirusResearch Institute, USA); derivatives of lipopolysaccharides such asmonophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton,Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide(t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OMPharma SA, Meyrin, Switzerland); and Leishmania elongation factor (apurified Leishmania protein; Corixa Corporation, Seattle, Wash.).

Adjuvants may be added to a composition comprising an immunogen, or, theadjuvant may be formulated with carriers, for example liposomes, ormetallic salts (e.g., aluminium salts (e.g., aluminium hydroxide)) priorto combining with or co-administration with a composition.

In some embodiments, a composition comprising an immunogen comprises asingle adjuvant. In other embodiments, a composition comprises two ormore adjuvants (See, e.g., WO 94/00153; WO 95/17210; WO 96/33739; WO98/56414; WO 99/12565; WO 99/11241; and WO 94/00153, each of which ishereby incorporated by reference in its entirety).

In some embodiments, a composition of the present invention may comprisesterile aqueous preparations. Acceptable vehicles and solvents include,but are not limited to, water, Ringer's solution, phosphate bufferedsaline and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose any bland fixed mineral or non-mineral oil maybe employed including synthetic mono-ordi-glycerides. In addition, fattyacids such as oleic acid find use in the preparation of injectables.Carrier formulations suitable for mucosal, subcutaneous, intramuscular,intraperitoneal, intravenous, or administration via other routes may befound in Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa.

A composition comprising an immunogen of the present invention can beused therapeutically (e.g., to enhance an immune response) or as aprophylactic (e.g., for immunization (e.g., to prevent signs or symptomsof disease)). A composition comprising an immunogen of the presentinvention can be administered to a subject via a number of differentdelivery routes and methods.

For example, the compositions of the present invention can beadministered to a subject (e.g., injection, orally, bath, or dip) bymultiple methods, including, but not limited to, those described herein.In some embodiments, a composition comprising an immunogen of thepresent invention may be used to protect or treat a fish susceptible to,or suffering from, disease by means of administering a composition ofthe present invention via a mucosal route (e.g., an oral route). Thus,the vaccine can be administered by any suitable known method ofinoculating fish including but not limited to immersion, oraladministration, spraying and injection. Preferably, the vaccine isadministered by mass administration techniques such as immersion asconducted by a standardized immersion protocol described by McAllisterand Owens (1986), the contents of which are hereby incorporated byreference herein in its entirety. When administered by injection, thevaccines are preferably administered parenterally. Parenteraladministration as used herein means administration by intravenous,subcutaneous, intramuscular, or intraperitoneal injection. Furtheradministration may be accomplished by sonification or electroporation.

Thus, in some embodiments, a composition comprising an immunogen of thepresent invention may be used to protect and/or treat a subjectsusceptible to, or suffering from, a disease by means of administeringthe composition by mucosal, intramuscular, intraperitoneal, intradermal,transdermal, pulmonary, intravenous, subcutaneous or other route ofadministration described herein. Methods of systemic administration ofthe vaccine preparations may include conventional syringes and needles,or devices designed for ballistic delivery of solid vaccines (See, e.g.,WO 99/27961, hereby incorporated by reference), or needleless pressureliquid jet device (See, e.g., U.S. Pat. No. 4,596,556; U.S. Pat. No.5,993,412, each of which are hereby incorporated by reference), ortransdermal patches (See, e.g., WO 97/48440; WO 98/28037, each of whichare hereby incorporated by reference). The present invention may also beused to enhance the immunogenicity of antigens applied to the skin(transdermal or transcutaneous delivery, See, e.g., WO 98/20734; WO98/28037, each of which are hereby incorporated by reference). Thus, insome embodiments, the present invention provides a delivery device forsystemic administration, pre-filled with the vaccine composition of thepresent invention.

In some embodiments, vaccine compositions are co-administered with oneor more antibiotics. For example, one or more antibiotics may beadministered with, before and/or after administration of thecomposition. The present invention is not limited by the type ofantibiotic co-administered. Indeed, a variety of antibiotics may beco-administered including, but not limited to, β-lactam antibiotics,penicillins (such as natural penicillins, aminopenicillins,penicillinase-resistant penicillins, carboxy penicillins, ureidopenicillins), cephalosporins (first generation, second generation, andthird generation cephalosporins), and other β-lactams (such as imipenem,monobactams,), β-lactamase inhibitors, vancomycin, aminoglycosides andspectinomycin, tetracyclines, chloramphenicol, erythromycin, lincomycin,clindamycin, rifampin, metronidazole, polymyxins, doxycycline,quinolones (e.g., ciprofloxacin), sulfonamides, trimethoprim, andquinolines.

There are an enormous amount of antimicrobial agents currently availablefor use in treating bacterial, fungal and viral infections. For acomprehensive treatise on the general classes of such drugs and theirmechanisms of action, the skilled artisan is referred to Goodman &Gilman's “The Pharmacological Basis of Therapeutics” Eds. Hardman etal., 9th Edition, Pub. McGraw Hill, chapters 43 through 50, 1996,(herein incorporated by reference in its entirety). Generally, theseagents include agents that inhibit cell wall synthesis (e.g.,penicillins, cephalosporins, cycloserine, vancomycin, bacitracin); andthe imidazole antifungal agents (e.g., miconazole, ketoconazole andclotrimazole); agents that act directly to disrupt the cell membrane ofthe microorganism (e.g., detergents such as polmyxin and colistimethateand the antifungals nystatin and amphotericin B); agents that affect theribosomal subunits to inhibit protein synthesis (e.g., chloramphenicol,the tetracyclines, erthromycin and clindamycin); agents that alterprotein synthesis and lead to cell death (e.g., aminoglycosides); agentsthat affect nucleic acid metabolism (e.g., the rifamycins and thequinolones); the antimetabolites (e.g., trimethoprim and sulfonamides);and the nucleic acid analogues such as zidovudine, gangcyclovir,vidarabine, and acyclovir which act to inhibit viral enzymes essentialfor DNA synthesis. Various combinations of antimicrobials may beemployed.

In some embodiments, a vaccine composition of the present invention isformulated in a concentrated dose that can be diluted prior toadministration to a fish. For example, dilutions of a concentratedcomposition may be administered to a subject such that the subjectreceives any one or more of the specific dosages provided herein. Insome embodiments, dilution of a concentrated composition may be madesuch that a fish is administered (e.g., in a single dose) a compositioncomprising 0.5-50% of the amount present in the concentratedcomposition. In some embodiments, a composition comprising an immunogenof the present invention (e.g., a concentrated composition) is stable atroom temperature for more than 1 week, in some embodiments for more than2 weeks, in some embodiments for more than 3 weeks, in some embodimentsfor more than 4 weeks, in some embodiments for more than 5 weeks, and insome embodiments for more than 6 weeks.

In some embodiments, following an initial administration of acomposition of the present invention (e.g., an initial vaccination), afish may receive one or more boost administrations (e.g., around 2weeks, around 3 weeks, around 4 weeks, around 5 weeks, around 6 weeks,around 7 weeks, around 8 weeks, around 10 weeks, around 3 months, around4 months, around 6 months, around 9 months, around 1 year, around 2years, around 3 years, around 5 years, around 10 years) subsequent to afirst, second, third, fourth, fifth, sixth, seventh, eighth, ninth,tenth, and/or more than tenth administration.

Dosage units may be proportionately increased or decreased based onseveral factors including, but not limited to, the weight of the fishand the life cycle of the fish. In addition, dosage units may beincreased or decreased for subsequent administrations (e.g., boostadministrations).

The vaccine may be stored in a sealed vial, ampule or the like. Thepresent vaccine can generally be administered in the form of a spray forintranasal administration, or by nose drops, inhalants, swabs ontonsils, or a capsule, liquid, suspension or elixirs for oraladministration. In the case where the vaccine is in a dried form, thevaccine is preferably dissolved or suspended in sterilized distilledwater before administration. Any inert carrier is preferably used, suchas saline, phosphate buffered saline, or any such carrier which thevaccine has suitable solubility. The compositions of the invention canbe administered to subjects in a biologically compatible form suitablefor pharmaceutical administration in vivo. By “biologically compatibleform suitable for administration in vivo” is meant a form of thecomposition to be administered in which any toxic effects are outweighedby the therapeutic effects of the membrane vesicles.

The composition may be administered in a convenient manner such as byinjection (subcutaneous, intravenous, etc.), oral administrationinhalation, transdermal application, or rectal administration. Thepharmaceutical compositions are therefore in solid or semisolid form,for example pills, tablets, creams, gelatin capsules, capsules,suppositories, soft gelatin capsules, gels, membranes, tubelets. Forparenteral and intracerebral uses, those forms for intramuscular orsubcutaneous administration can be used, or forms for infusion orintravenous or intracerebral injection can be used, and can therefore beprepared as solutions of the active membrane vesicles or as powders ofthe vesicles to be mixed with one or more pharmaceutically acceptableexcipients or diluents, suitable for the aforesaid uses and with anosmolarity which is compatible with the physiological fluids. For localuse, those preparations in the form of creams or ointments for topicaluse, or in the form of sprays are suitable; for inhalant uses,preparations in the form of sprays, for example nose sprays, aresuitable.

It is contemplated that the compositions and methods of the presentinvention will find use in various settings, including research,aquaculture (e.g., for food), the wild, ornamental fish, etc. Forexample, compositions and methods of the present invention also find usein studies of the immune system of fish. In some embodiments, thevaccine compositions find use in commercial settings (e.g., commercialfish farming). The vaccines find use in immunizing a variety of speciesof fish. Examples include, but are not limited to, Atlantic cod, Gadusmorhua; tilapia, Oreochromis sp.; Atlantic salmon, Salmo solar; hybridstriped bass, Morone chrysops×M. saxatilis and three-lined grunt,Parapristipoma trilinineatum.

The present invention further provides kits comprising the vaccinecompositions comprised herein. In some embodiments, the kit includes allof the components necessary, sufficient or useful for administering thevaccine. For example, in some embodiments, the kits comprise devices foradministering the vaccine (e.g., needles or other injection devices),temperature control components (e.g., refrigeration or other coolingcomponents), sanitation components (e.g., alcohol swabs for sanitizingthe site of injection) and instructions for administering the vaccine.

Example 1 Materials & Methods

Strains, Media and Growth Conditions.

F. noatunensis ssp. noatunensis NCIMB14265 isolated from diseasedAtlantic cod Gadus morhua L. in Norway (Ottem et al., 2007) and F.philomiragia ssp. philomiragia ATCC25015 was kept for long term storageat −80° C. as previously described. Cultivation of bacteria on solidmedia was performed on ECA plates without antibiotics at 20-22° C., andliquid cultures were performed in Eugon Broth supplemented with 2 mMFeCl₃ as previously described (Brudal et al., 2013).

Isolation of OMVs.

For initial experiments, 10 ml overnight cultures were used to inoculate100 ml liquid cultures until OD₆₀₀≈0.1, and grown to late-logarithmic orearly stationary growth phase. The bacteria were pelleted at 15 000 g at4° C. for 10 minutes, and the supernatant harvested. OMV-containingsupernatant was sterile filtered through 0.45 μm filters, followed by asecond filtration step using 0.2 μm filters to remove any contaminatingcells and cell debris. 70 ml double-filtered OMV-containing supernatantwas subjected to ultracentrifugation at 125 000 g at 4° C. using aOptima LE-80K Ultracentrifuge (Beckman Instruments) for 2 hours topellet OMVs. The supernatant was removed, the pellet resuspended in 50mM Hepes buffer pH 6.8 and OMVs were re-pelleted by a secondcentrifugation at 125 000 g for 30 minutes. The supernatant was removed,and the pellet resuspended in 100 μl PBS pH 7.4. Protein concentrationwas measured by NanoDrop. 25 μl aliquots were stored at −80° C. forlong-term storage, and one aliquot streaked on an ECA plate andincubated at 20-22° C. for at least three weeks to ensure sterility. Forlarge-scale production of OMVs, 2×350 ml culture was used, and a totalvolume of 420 ml double-filtered OMV containing supernatant was used forisolation.

SDS-PAGE and GC-MS.

Aliquots of OMVs were separated by SDS-PAGE followed by staining withCoomassie Blue. The major bands of interest were cut from the gel with aclean scalpel, and stored in individual eppendorf tubes at 4° C. untilfurther processing. For preparation for GC-MS, the gel pieces werewashed with HPLC water for 15 minutes, and washed twice with 50%acetonitrile (ACN) in HPLC water for 20 minutes each time. Thereafter,the gel pieces were dehydrated in 100% ACN overnight. The supernatantwas discarded, the gel pieces rehydrated in 50 mM ammonium bicarbonatein HPLC water (Abb) with 3.0 mg/ml dithiothreitol and incubated at 56°C. for one hour to reduce intermolecular disulfide bonds in proteins.Subsequently, the supernatant was discarded and the samples rehydratedwith 50 mM Abb containing 10 mg/ml iodoacetamide and incubated at roomtemperature in the dark for 45 minutes for alkylation of proteins toprevent the construction of new disfulfde-bonds. The supernatant wasdiscarded, and the samples washed three times with 50% ACN in HPLC waterand dehydrated in 100% ACN. Thereafter, the samples were trypsinatedwith 16 ng/μl Trypsin in 50 mM Abb at 37° C. overnight. Trypsinatedpeptides were isolated from the gel pieces by addition of 5% formic acid(FA) in HPLC water and the supernatant removed, followed by 2 treatmentswith 5% FA in 50% ACN and complete dehydration in 100% ACN. Therecovered protein-containing supernants were dried using Techne SampleConcentrator. The samples were resuspended in 1% FA, and solid phaseextraction was performed with a C18 filter to clean up the samples.Finally, the samples were again dehydrated using Techne SampleConcentrator, and stored at −20° C. until further analysis.

Atomic Force Imaging of OMVs.

For Atomic force imaging (AFM), MgCl₂ was added to a final concentrationof 10 mM to an aliquot of isolated OMVs, and 10 μl of the suspension wasapplied to a freshly cleaved mica surface. The OMVs were allowed toadhere to the surface for 10 minutes before washing the surface 8 timeswith 100 μl MQ water. Excess water was removed, and the specimencarefully dried with N₂-gas. Images were recorded inintermittent-contact mode at room temperature using a NanoWizardMicroscope (JPK Instruments AG, Berlin, Germany) with a scan frequencyof 1.0 Hz using ultrasharp silicon cantilevers with silicon etched probetips, NSC35/AlBS (MikroMasch, Madrid, Spain). AFM images were analyzedusing The NanoWizard® IP Image Processing Software (JPK Instruments AG).The theoretical size of the OMVs were calculated according to Pierson etal. (2011), in short we assumed that when OMVs adhere to the micasurface they assume the shape of half a sphere, calculated the volume ofthat half sphere based on V= 4/3πabc/2 and then use the calculatedvolume to determine the diameter of a perfect sphere (the correctdiameter of the OMV). For calculation of nanotubes we assumed that whennanotubes adhere to mica they assume the volume of half a cylinder,calculated the volume of half a cylinder based on V=πr²h/2 and used thecalculated volume to determine what size the nanotube would be as aperfect sphere.

Transmission Electron Microscopy.

Carbon-coated grids were pretreated with poly-L-lysine for 20 minutesand washed three times with MQ water. Thereafter, one aliquot of OMVswere allowed to adhere for 10 minutes before the grids were washed threetimes with PBS, two times with MQ water, stained for 1 minute with 4%uranylacatate and washed once with MQ water. The grids were analyzedwith a microscope.

Preparation of Bacterial Cultures for Zebrafish Infection.

Preparation of bacterial suspensions for infection experiments andcalculation of CFU was performed essentially as described previously(Brudal et al., submitted). In short, F. noatunensis ssp. noatunensiswas cultured in EBF, cells collected by centrifugation, resuspended inPBS pH 7.4 and OD₆₀₀ adjusted to the desired concentration. Serialdilutions was performed and plated on ECA plates for CFU estimation.

Zebrafish Embryo Infection Trial.

Infection of zebrafish embryos were performed essentially as previouslydescribed (Brudal et al., submitted). In short, 15 Zebrafish embryo ABwt were intravascularly injected with 9×10^3 CFU of F. noatunensis ssp.noatunensis pkk289KM/GFP wt and observed for a period of 7 days. At 7dpi, the embryos were euthanized and fixed in 10% buffered formalin atroom temperature for 24 hours to ensure proper penetration of thefixative into the tissues, and thereafter stored at 4° C. until furtherprocessing. Whole zebrafish embryos were fixed.

F. noatunensis ssp. noatunensis Dose-Response Experiment in Zebrafish.

Male and female Zebrafish Danio rerio L. wild type strain AB 10 monthsof age were obtained from the Aleström Zebrafish Lab facility at theNorwegian School for Veterinary Sciences and kept in 6 liter sized fishtanks Fish were fed daily with SDS 400 Scientific Fish Food. Fish waterwas made by The fish were kept at room temperature (20+−2° C.) andacclimatized for at least one week prior to injections. 50% water waschanged twice daily, and the water was aerated using a (mm diameterhose). The following water parameters were monitored using commercialtest kits (TetraTest kit): water hardness (KH and GH), pH, NO₂ ⁻, NO₃²⁻, NH₃/NH₄ ⁺ and O₂. Three groups of 15 fish each were used for thedose-response experiment. The fish were anesthetized with Tricainemethanesulfonate (MS-222, Sigma-Aldrich) 100 mg/L, transferred to apresoaked sponge with grooves cut into it to keep the fish in place withthe abdomen facing upwards and injected in the intraperitoneal cavity(i.p.) with a 30 G 0.3 mm×8 mm Micro-Fine Demi needle with syringe.After infection, the fish were immediately transferred to a recoverybath before being transferred back to the holding tanks. The first groupwas injected with 25 μl of F. noatunensis ssp. noatunensis OD₆₀₀ in PBS20.0 (10⁹ cfu), the second group was injected i.p. with 25 μl of F.noatunensis ssp. noatunensis OD₆₀₀ in PBS 2.0 (10^8 cfu) and the thirdgroup injected i.p. with 25 μl PBS. Mortality was recorded twice daily,and moribound fish were euthanized with 300 mg/L Tricainemethanesulfonate due to ethical considerations.

Immunization of Zebrafish with OMVs.

Three tanks with 10 month old zebrafish AB wt, 18 fish in each tank,were acclimatized for 2 weeks prior to immunization experiments. 2groups were anesthetized as previously described and vaccinated i.p.with 40 μg OMVs in 25 μl PBS, while the third group was mock-vaccinatedwith 25 μl PBS. One month later (637 degree-days) one OMV vaccinated andthe PBS mock-vaccinated group were injected i.p. with 25 μl F.noatunensis ssp. noatunensis OD₆₀₀ in PBS 2.0 (10⁸ CFU), while theremaining OMV vaccinated group was mock-infected i.p. with 25 μl PBS.

Quantification of Bacterial Burden.

For the dose-response experiment, three randomly chosen fish from eachgroup were euthanized as previously described at each time point. Forthe vaccination experiment, the number of fish at each time point was 4.Necropsy was performed on all euthanized fish, and the spleen, heart andkidney harvested, transferred to RNAlater (Ambion) and stored at 4° C.until further processing. RNAlater was removed, and the samplestransferred to 2.0 ml SafeLock Eppendorf tubes containing 100 μl lysisbuffer with 20 mg/ml lysozyme (Sigma-Aldrich) and a 0.5 mm diameterstainless steel bead (QIAGEN). The tissue was homogenized by TissueLyserII at 15 Hz for 20 seconds, and genomic DNA (gDNA) extracted with theQIAGEN DNEasy Blood & Tissue Mini kit according to the manufacturersinstructions. 100 μl DEPC treated H₂O was used for elution of gDNA, and10 μl of the eluate was diluted 1:10 in DEPC H₂O and used as templatefor qPCR.

The previously published diagnostically validated primer pair targetinga hypothetical protein in F. noatunensis ssp. noatunensis with accessionnumber JQ780324 (Duodu et al., 2013) was chosen for absolutequantification of the amount of F. noatunensis ssp. noatunensis genomeequivalents (GE) in each fish tissue. Quantitative PCR was performed intriplicates using Express SYBR GreenER qPCR Supermix Universal (LifeTechnologies Inc.), 50 μM Rox Reference dye, 300 μM forward and reverseprimers, and 5 μl template (corresponding to 1/200 of the total amountof extracted gDNA for each well) and a Stratagene Mx3005p qPCR machine.The qPCR reaction conditions were as followed: 2 minutes at 50° C., 2minutes at 95° C., followed by 40 cycles 15 seconds at 95° C. and 1minute at 60° C. Melting curve analysis of the PCR product was performedto verify single amplification peaks. The primer binding efficiency wasvalidated using serial 10-fold dilutions of gDNA isolated from zebrafishtissue as described by Duodu et al. (2013), and similar results wereobtained as Duodu et al (2013) obtained from Atlantic cod tissue with F.noatunensis ssp. noatunensis. The calculated primer binding efficiencywas used to estimate the relative amount of F. noatunensis ssp.noatunensis GE compared to 1 ng of F. noatunensis ssp. noatunensis gDNAused as equilibrator on each qPCR plate, and absolute quantification wasperformed under the assumption that 20 fg gDNA corresponds to 10 GE forF. noatunensis ssp. noatunensis (Duodu et al., 2013).

Statistical Analysis.

Statistical analysis of the data sets was performed using JMP 8.0.2.(SAS Institute Inc., Cary, N.C., USA). Differences in bacterialquantification between groups were deemed statistically significant ifp<0.05 using a one-tailed Student's t-test assuming unequal variance.Kaplan-Meier survival analysis (Goel et al., 2010) was used foranalyzing survival, and differences between groups were deemedstatistically significant if p-value <0.05 using Wilcoxon-test andLog-rank test.

Results

F. noatunensis ssp. noatunensis Produces Large Amounts of OMVs in LiquidCulture.

From 420 ml of late-log/early stationary liquid culture of F.noatunensis ssp. noatunensis, an average of 1860 μg protein wasrecovered. This corresponds to 2.66 μg protein per ml culture. For F.philomiragia ssp. philomiragia the corresponding value was 0.06 μgprotein per ml culture. We did not investigate what culture conditionswould give the optimal OMV production for F. philomiragia ssp.philomiragia. Pierson et al. (2011) did this for F. tularensis ssp.novicida and got a 86 μg OMV protein yield from 350 ml culture underoptimal conditions.

OMVs Contain Numerous Proteins, Some of which are Associated withVirulence.

OMVs isolated from F. noatunensis ssp. noatunensis contain abundantproteins easily detectable by Coomassie Blue staining. GC-MS analysis ofthe most abundant proteins identified three proteins associated with theFrancisella pathogenicity island (FPI), the major outer membrane protein(FopA) and a chaperonin (GroEL). The most abundant protein is IglC,followed by PdpD and PdpA.

OMVs are Isolated Intact.

To verify that OMVs were isolated intact, and that they were in theexpected size range for OMVs, we performed AFM and TEM on isolated OMVs.Numerous OMVs of spherical shape were visible by AFM, though only a fewnanotubes (McCaig et al., 2013) could be observed. The mean diameter ofOMVs from F. noatunensis ssp. noatunensis was 72.34 nm (SD=26.17), themedian diameter was 67.32 nm, and the measured size range was 24-133 nm.This corresponds well with the expected size range for OMVs (10-300 nmin diameter), and with data published by Pierson et al. (2011) for OMVsfrom F. tularensis ssp. novicida (97 nm) and F. philomiragia ssp.philomiragia (76 nm). Surprisingly, thin appendages were clearly visibleprotruding from many of the vesicles, sometimes connecting severalvesicles together in a meshwork. These did not appear to be similar tothe nanotubes described by McCaig et al. (2013), as they were only 0.5-1nm in height and approximately 25 nm in width. Observed nanotubes on AFMwere measured as 800-1000 nm in length, 11-18 nm in height and a widthof 100-120 nm, and would have a mean diameter of 50.7 nm as perfectspheres, supporting a hypothesis that nanotubes and OMVs could be thesame biological structures, possibly observed in different stages ofdevelopment. On TEM images, numerous nanotubes in addition to OMVs wereobserved. The difference in ratio between OMVs and nanotubes on AFM andTEM might be due to difference in the ability of nanotubes to adhere tothe mica surface used for AFM sample processing.

OMVs are Produced In Vivo in Infected Zebrafish Embryos.

TEM images of zebrafish embryos infected with F.n.n.-GFP verifiedprevious observations by fluorescence microscopy (Brudal et al.,):bacteria were present both intracellular in infected cells and in theextracellular milieu. Some host cells (macrophages) were heavilyinfected and were in various stages of cell death, and while we couldfind bacteria intracellularly in other cell types as well (such asendothelial cells) these did not appear to be dying. OMVs could beobserved in the near vicinity of bacteria in infected host tissues, andin rare events OMVs could be observed budding from the bacteria.

F. noatunensis ssp. noatunensis Causes Dose-Dependent Mortality of AdultZebrafish.

Onset of mortality for zebrafish that were infected with the highestdose (10⁹ cfu) occurred at 2 dpi, while there was an initial delay inonset of mortality for the group infected with 10⁸ cfu, starting at 4dpi. No mortality was observed in the PBS injected controls. Allinfected fish exhibited decreased appetite and decreased motility, andmoribound fish had erratic swimming behavior. At 14 dpi, all remainingfish in the high infection group (n=3) were sampled for quantificationof bacterial burden, and that group was therefore terminated. Mortalityin the 10^8 cfu infected group was observed on day 4 and 9, andthereafter no mortality was observed until the end of the observationperiod at 28 dpi. Significant difference in survival between groupsinfected with high and low dose was observed (p-value 0.0142 wilcoxontest, p-value 0.0142 log-rank test).

OMvs are Safe for Immunization of Zebrafish, and Protects fromSubsequent Challenge with F. noatunensis ssp. noatunensis.

One fish vaccinated with OMVs developed problems with keeping afloat inthe water and was euthanized. Upon necropsy, inflammation and deflationof the anterior segment of the swim bladder was evident, probably causedby puncture of this site due to an unfortunate injection. No otherevidence of discomfort due to vaccination was observed, the fish wereeating and behaving normally from the first day after vaccination. Afterinfection, initially one fish died in the group vaccinated with OMVs andinfected with 10⁸ cfu F. noatunensis ssp. noatunensis in the first day.This might have been due to damage from the injection, as the fish wasquite pale upon necropsy. Unvaccinated+infected fish exhibited anorexiaand decreased motility as in the first experiment for the first twoweeks of the experiment, while the vaccinated+infected fish had slightdecrease in appetite the first couple of days, but much less reducedcompared to the unvaccinated group and was undistinguishable from thecontrol group at 4 dpi. The vaccinated+uninfected group exhibited noclinical symptoms throughout the experiment.

Quantification of bacterial burden showed approximately a 11 fold higherbacterial burden in the kidney of unvaccinated compared to vaccinatedfish throughout the experiment (p-value 0.0239), while the correspondingnumbers for spleen and kidney was 5-fold (p-value 0.0379) and 4-fold(p-value 0.0949) respectively. The relative amount of GE betweenvaccinated and unvaccinated fish in each tissue type was quite stablefor all tissues regardless of time point examined, while the absolutequantification of GE was at the highest level at the first time pointexamined (1 week) and declined during the course of infection.

Example 2. Liquid Culture Growth of P. SALMONIS to High Densities

Materials & Methods

Strains, media and growth conditions. P. salmonis was kept for long termstorage at −80° C. in 10% skimmed milk or in Eugon broth supplementedwith 20% glycerol. Cultivation of bacteria on solid media was performedon ECA plates, and liquid cultures were performed in Eugon Brothsupplemented with 0.1% Casamino acid (BD suppliers).

OMV Isolation From P. salmonis.

2×10 ml overnight cultures were used to inoculate 2×100 ml liquidcultures of P. salmonis in Eugon broth supplemented with Casamino acidsand grown overnight to mid-logarithmic growth phase. The bacteria werepelleted at 15 000 g at 4° C. for 10 minutes, and the supernatantharvested. OMV-containing supernatant was sterile filtered through 0.45μm filters, followed by a second filtration step using 0.2 μm filters toremove any contaminating cells and cell debris. 140 ml double-filteredOMV-containing supernatant was subjected to ultracentrifugation at 125000 g at 4° C. using a Optima LE-80K Ultracentrifuge (BeckmanInstruments) for 2 hours to pellet OMVs. The supernatant was removed,the pellet resuspended in 50 mM Hepes buffer pH 6.8 and OMVs werere-pelleted by a second centrifugation at 125 000 g for 30 minutes. Thesupernatant was removed, and the pellet resuspended in 100 μl PBS pH7.4. Protein concentration was measured by NanoDrop. 25 μl aliquots werestored at −80° C. for long-term storage, and one aliquot streaked on anECA plate and incubated at 20-22° C. for at least three weeks to ensuresterility.

Initial Characterization of P. salmonis OMVs

2-fold dilution series of OMVs isolated from P. salmonis were subjectedto SDS-PAGE followed by Coomassie staining to identify the major proteincontent. Atomic force microscopy imaging was performed to verify that P.salmonis OMVs were isolated intact, essentially as described for F.noatunensis OMVs. MgCl₂ was added to a final concentration of 10 mM toan aliquot of isolated OMVs, and 10 μl of the suspension was applied toa freshly cleaved mica surface. The OMVs were allowed to adhere to thesurface for 10 minutes before washing the surface 8 times with 100 μl MQwater. Excess water was removed, and the specimen carefully dried withN₂-gas. Images were recorded in intermittent-contact mode at roomtemperature using a NanoWizard Microscope (JPK Instruments AG, Berlin,Germany) with a scan frequency of 1.0 Hz using ultrasharp siliconcantilevers with silicon etched probe tips, NSC35/AlBS (MikroMasch,Madrid, Spain). AFM images were analyzed using The Nano Wizard® IP ImageProcessing Software (JPK Instruments AG). The theoretical size of theOMVs were calculated according to Pierson et al. (2011), in short weassumed that when OMVs adhere to the mica surface they assume the shapeof half a sphere, calculated the volume of that half sphere based onV=4/3πabc/2 and then use the calculated volume to determine the diameterof a perfect sphere (the correct diameter of the OMV).

Results

Eugon broth supplemented with casamino acids supported the growth of P.salmonis to high optical densities. OMVs isolated from mid-logarithmiccultures yielded 128.3 μg OMV proteins from 140 ml double-filteredOMV-containing supernatant, a yield of 0.92 μg pr ml culture. Thedominating P. salmonis OMV protein as evaluated by Coomassie bluestaining had an apparent molecular weight of approximately 47 kDa, butseveral additional distinct bands of lower and higher molecular weightwere also present in the sample. AFM imaging verified that OMVs wereisolated intact. The mean calculated diameter of P. salmonis OMVs was47.6 nm.

Example 3. Immune Response of Adult Zebrafish Against High (1×10¹⁰ andLower (1×10⁷) Dose of P. Salmonis and Against Exposure of OMV Isolatedfrom P. SALMONIS

Adult zebrafish were infected with 1×10¹⁰ or 1×10⁷ CFU of P. salmonis.Fish infected with 1010 CFU started to die after 3 days, while fishinfected with 107 CFU started to die after 5 days. 50% of the fish inboth groups were dead after 6 days.

Adult zebrafish were then injected with 40 ug OMV isolated from P.salmonis. No toxicity was observed after 7 days.

Immune responses were analyzed in the spleen, heart, kidney and gills ofzebrafish infected with P. salmonis (FIGS. 10 a, b, c and d and 11 a, b,c and d) and injected with OMVs isolated from P. salmonis (FIGS. 12 a,b, c and d). The zebrafish infected with P. salmonis strain 5692 inducedan overall high pro-inflammatory immune response with tnfa suggesting ahigh initial response to the infection. Similar responses have beendetected in Atlantic salmon infected with P. salmonis (Tacchi et al.,Physiological Genomics, 2011, 43:21 1241-54). Here, the response wasreduced in the 10⁷ challenge dose compared to the higher 10¹⁰ challengedose suggesting a dose-response effect of the pathogen in the infectionmodel. The high expression in the kidney of most immune genes tested,including il8, could be explained by the fact that the kidney is a majorimmune organ in fish and early immune responses are often found in thistissue. Many pathogens including Francisella utilize the suppression ofcytokine signaling (SOCS) pathways to inhibit the host's ability toclear an infection (Brudal et al., 2014). Interestingly, the effect ofzebrafish infection with P. salmonis does not increase in transcriptionof the soc3b gene, except for the very high dose 10¹⁰ in the kidneysuggesting the P. salmonis does not suppress cytokine signaling in thesame way as Francisella infections in the zebrafish. This is supportedwith the high expression of the cytokines IL8 and INFγ with the P.salmonis infection. Although the zebrafish immune response was reducedin the infection with the 5892 strain isolated from Atlantic salmon fromCanada, the overall induction of the immune genes were similar to thosedetected for strain 5692. Clearly these results taken together supportthe zebrafish as a good infection model that inducing similar immuneresponse as the Atlantic salmon specific for P. salmonis infection.

Injection with the isolated OMV form P. salmonis strain 5692 induced lowimmune responses compared to the high doses for the bacterialinjections. This supports our previous results that OMV are not toxicfor the host. Still, the OMV modulate an immune response that isdifferent from the infection and the PBS control which is important forvaccine function.

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All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled in therelevant fields are intended to be within the scope of the followingclaims.

The invention claimed is:
 1. A vaccine for providing immunity in a fishagainst microorganisms comprising: a protective amount of a purifiedpreparation of outer membrane vesicles of a microorganism and aphysiologically acceptable carrier for a fish, wherein the microorganismis Piscirickettsia ssp.
 2. The vaccine of claim 1, wherein saidPiscirickettsia spp. is selected from the group consisting ofPiscirickettsia salmonis strains LF-89; (U36941); EM-90 (U36940); NOR-92(U36942); ATL-4-91 (U36915); IRE-99D (AY498637); SCO-95A (AY498636);SCO-02A (AY498635); IRE-98A (AY498634); IRE-91A (AY498633); WSB-98; andAL10005.
 3. The vaccine of claim 1, wherein said fish is selected fromthe group consisting of Atlantic cod, Gadus morhua; tilapia, Oreochromissp.; Atlantic salmon, Salmo salar; hybrid striped bass, Moronechrysops×M. saxatilis and three-lined grunt, Parapristipomatrilinineatum.
 4. The vaccine of claim 1, wherein said vaccine protectssaid fish against Salmon Rickettsial Septicaemia (SRS).